The present invention relates to methods for detecting and mapping genetic abnormalities associated with various diseases. In particular, it relates to the use of nucleic acid hybridization methods for comparing copy numbers of particular nucleic acid sequences in a collection of sequences relative to the copy number of these sequences in other collections of sequences.
Many genomic and genetic studies are directed to the identification of differences in gene dosage or expression among cell populations for the study and detection of disease. For example, many malignancies involve the gain or loss of DNA sequences resulting in activation of oncogenes or inactivation of tumor suppressor genes. Identification of the genetic events leading to neoplastic transformation and subsequent progression can facilitate efforts to define the biological basis for disease, improve prognostication of therapeutic response, and permit earlier tumor detection.
In addition, perinatal genetic problems frequently result from loss or gain of chromosome segments such as trisomy 21 or the micro deletion syndromes. Thus, methods of prenatal detection of such abnormalities can be helpful in early diagnosis of disease.
Cytogenetics is the traditional method for detecting amplified or deleted chromosomal regions. The resolution of cytogenetic techniques is limited, however, to regions larger than approximately 10 Mb (approximately the width of a band in Giemsa-stained chromosomes). In complex karyotypes with multiple translocations and other genetic changes, traditional cytogenetic analysis is of little utility because karyotype information cannot be fully interpreted. Furthermore conventional cytogenetic banding analysis is time consuming, labor intensive, and frequently difficult or impossible due to difficulties in obtaining adequate metaphase chromosomes. In addition, the cytogenetic signatures of gene amplification, homogeneously staining regions (HSR), or double minute chromosomes, do not provide any information that contributes to the identification of the sequences that are amplified.
More recent methods permit assessing the amount of a given nucleic acid sequence in a sample using molecular techniques. These methods (e.g., Southern blotting) employ cloned DNA or RNA probes that are hybridized to isolated DNA. Southern blotting and related techniques are effective even if the genome is heavily rearranged so as to eliminate useful karyotype information. However, these methods require use of a probe specific for the sequence to be analyzed. Thus, it is necessary to employ very many individual probes, one at a time, to survey the entire genome of each specimen, if no prior information on particular suspect regions of the genome is available.
Comparative genomic hybridization (CGH) is a more recent approach to detect the presence and identify the location of amplified or deleted sequences. See, Kallioniemi et al., Science 258: 818-821 (1992) and WO 93/18186). CGH reveals increases and decreases irrespective of genome rearrangement. In one implementation of CGH, genomic DNA is isolated from normal reference cells, as well as from test cells (e.g., tumor cells). The two nucleic acids are differentially labeled and then hybridized in situ to metaphase chromosomes of a reference cell. The repetitive sequences in both the reference and test DNAs are either removed or their hybridization capacity is reduced by some means. Chromosomal regions in the test cells which are at increased or decreased copy number can be quickly identified by detecting-regions where the ratio of signal from the two DNAs is altered. For example, those regions that have been decreased in copy number in the test cells will show relatively lower signal from the test DNA than the reference compared to other regions of the genome. Regions that have been increased in copy number in the test cells will show relatively higher signal from the test DNA.
Thus, CGH discovers and maps the location of the sequences with variant copy number without prior knowledge of the sequences. No probes for specific sequences are required and only a single hybridization is required. Where a decrease or an increase in copy number is limited to the loss or gain of one copy of a sequence, the CGH resolution is usually about 5-10 Mb.
New techniques which provide increased sensitivity, more precise localization of chromosomal abnormalities and which can detect differences in levels of gene expression are particularly desirable for the diagnosis of disease. The present invention provides these and other benefits.